Effects of beta-amyloid (25-35) on learning in the common snail.

The effects of neurotoxic beta-amyloid fragment (25-35) on the formation of behavioral sensitization and a conditioned defensive reflex to food were studied. Administration of beta-amyloid (25-35) to common snails before the start of training led to a significant reduction in sensitization of the defensive reaction, weakening of the formation of the conditioned defensive reflex to food, and impairment of memory. These impairments to behavioral plasticity may be mediated by changes in synaptic plasticity previously observed in the presence of beta-amyloid.

Cannabinoid regulation in identified synapse of terrestrial snail.

In the terrestrial snail a direct monosynaptic glutamatergic connection between the primary sensory neuron and a premotor interneuron involved in withdrawal behaviour can be functionally identified using electrophysiological techniques. We investigated the involvement of cannabinoids in regulation of this synaptic contact. The results demonstrate that the specific binding sites for agonists to mammalian type 1 cannabinoid receptors (CB1Rs) exist in the snail's nervous system. Application of a synthetic cannabinoid agonist anandamide selectively changed the efficacy of synaptic contacts between the identified neurons. A decrease in the long-term synaptic facilitation of the synaptic contact elicited by high-frequency nerve tetanization in the presence of cannabinoid agonist anandamide was observed, suggesting a possible role of endocannabinoids in regulation of plasticity at this synaptic site. The selective antagonist of CB1Rs [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] AM251 bath application was changing the efficacy of the synaptic contact only when the postsynaptic neuron had been intracellularly activated before its application. This observation implies an involvement of endocannabinoids in plasticity phenomena induced by activity in the postsynaptic target. Additional support of endocannabinoid involvement in synaptic function at this site was given by experiments in which AM251 blocked the short-term suppression of synaptic excitation evoked by low-frequency nerve tetanization, a phenomenon qualitatively similar to cannabinoid-dependent synaptically evoked suppression of excitation demonstrated in the mammalian nervous system. The results of the present study suggest an involvement of cannabinoids in the regulation of synaptic efficacy. Further, anandamide could be a candidate for an endogenous neuromessenger involved in plasticity processes.

[Beta-amyloid peptide influences behavioral plasticity in terrestrial snail].

Influence of neurotoxic fragment of beta-amyloid peptide (25-35) on Helix lucorum behavioral plasticity (sensitization and food-aversion learning) was investigated. After beta-amyloid peptide (25-35) injection a significant reduction of behavioral long-term sensitization was observed. It was found, that beta-amyloid peptide (25-35) may interfere with associative learning and memory. Our results clearly demonstrate that beta-amyloid peptide (25-35) may play a significant role in behavioral plasticity by chronically eliminating certain underlying forms of synaptic plasticity.

Caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail Helix.

Although caspase activity in the nervous system of mollusks has not been described before, we suggested that these cysteine proteases might be involved in the phenomena of neuroplasticity in mollusks. We directly measured caspase-3 (DEVDase) activity in the Helix lucorum central nervous system (CNS) using a fluorometrical approach and showed that the caspase-3-like immunoreactivity is present in the central neurons of Helix. Western blots revealed the presence of caspase-3-immunoreactive proteins with a molecular mass of 29 kDa. Staurosporin application, routinely used to induce apoptosis in mammalian neurons through the activating cleavage of caspase-3, did not result in the appearance of a smaller subunit corresponding to the active caspase in the snail. However, it did increase the enzyme activity in the snail CNS. This suggests differences in the regulation of caspase-3 activity in mammals and snails. In the snail CNS, the caspase homolog seems to possess an active center without activating cleavage typical for mammals. In electrophysiological experiments with identified snail neurons, selective blockade of the caspase-3 with the irreversible and cell-permeable inhibitor of caspase-3 N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp-(OMe)-fluoro-methylketone prevented development of the long-term stage of synaptic input sensitization, suggesting that caspase is necessary for normal synaptic plasticity in snails. The results of our study give the first direct evidence that the caspase-3-like activity is essential for long-term plasticity in the invertebrate neurons. This activity is presumably involved in removing inhibitory constraints on the storage of long-term memory.

Ephaptic feedback in identified synapses in mollusk neurons.

The possible existence of intrasynaptic ephaptic feedback in the invertebrate CNS was studied. Intracellular recordings were made of excitatory postsynaptic potentials and currents arising on activation of the recently described monosynaptic connection between identified neurons in the snail CNS. In the presence of ephaptic feedback, tetanization of the postsynaptic neuron with hyperpolarizing impulses should activate presynaptic calcium channels, thus increasing the amplitude of excitatory postsynaptic potential, while sufficiently strong postsynaptic hyperpolarization applied during generation of the excitatory postsynaptic current should induce "supralinear" increases in its amplitude, as has been observed previously in rat hippocampal neurons. The first series of experiments involved delivery of 10 trains of hyperpolarizing postsynaptic impulses (40-50 mV, duration 0.5 sec, frequency 1 Hz, train duration 45 sec); significant changes in the amplitude of excitatory postsynaptic were not seen. In the second series of experiments, changes in the amplitude of the excitatory postsynaptic current were studied during hyperpolarization of the postsynaptic neuron. At a potential of -100 mV, the amplitude of the excitatory postsynaptic current increased significantly more than predicted by its "classical" linear relationship with membrane potential. This "supralinear" increase in the amplitude of the excitatory postsynaptic potential can be explained by the operation of ephaptic feedback and is the first evidence for this phenomenon in CNS synapses of invertebrates.

[Ephaptic feedback in identified synapses of terrestrial snails]. / Efapticheskaia obratnaia sviaz' v identsifitsirovannom sinapse nazemnogo molliuska.

A hypothesis for the existence of the intrasynaptic ephaptic feedback (EFB) in the invertebrate central nervous sytem was tested. Excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) evoked by the activation of the recently described monosynaptic connection between the identified snail neurons were recorded intracellularly. In case of the EFB presence, the postsynaptic tetanization with hyperpolarization pulses could activate presynaptic Ca2+ channels and enhance the EPSP amplitude, whereas a steady postsynaptic hyperpolarization should induce a "supralinear" increase in EPSC amplitudes as it has been found in the rat hippocampus. In the first series of the experiments, 10 trains of hyperpolarizing pulses (40-50 mV, 1 Hz, pulse duration 0.5 s, train duration 45 s) were delivered postsynaptically. No significant changes in EPSP amplitudes were found. In the second series of the experiments, the EPSC amplitudes were measured during varying postsynaptic hyperpolarization. At the membrane potential 100 mV, the EPSP amplitude was significantly higher than theoretically predicted from the classical linear dependence. Such a "supralinear" effect of postsynaptic depolarization can be explained by the presence of the EFB. This finding is the first evidence for the EFB existence in the invertebrate central nervous system.

Postsynaptic calcium contributes to reinforcement in a three-neuron network exhibiting associative plasticity.

We show that activation of a single serotonergic cell is sufficient to trigger long-term associative enhancement of synaptic input to the withdrawal interneuron in a simple network consisting of three interconnected identified cells in the nervous system of terrestrial snail Helix. 1,2-bis (2-aminophenoxy) Ethane-N,N,N',N'-tetraacetic acid (BAPTA) injection in the postsynaptic neuron abolishes the pairing-specific enhancement of synaptic input. Activation of a single modulatory cell that we used to reinforce the synaptic input induced an increase of the intracellular [Ca2+] in the ipsilateral withdrawal interneuron without any changes of its membrane potential or input resistance. Similar changes in intracellular [Ca2+] were observed in the same withdrawal interneuron under bath application of 10(-5) m serotonin. Responses to repeated glutamate applications to the soma of synaptically isolated withdrawal interneurons increased after 10 min of serotonin or thapsigargin bath application, but were absent in conditions of preliminary BAPTA intracellular injection, significantly decreased under heparin injection. Thus, activity of a single modulatory cell may mediate reinforcement via an increase of [Ca2+] in the postsynaptic cell in a simple network consisting of neurons with defined behavioural roles.

Phase-dependent coordination of two motor programs in the buccal ganglion of a pteropod mollusk.

Rhythmic activity in two independent structures of the digestive apparatus of Clione limacina--the radula and the hooks--is coordinated by neural networks in the buccal ganglion during feeding behavior. Optical recording of neuron activity in the buccal ganglion, which allows simultaneous recording of large numbers of neurons, showed that the activity of all neurons producing volley discharges can be assigned to only two phases of a single rhythm. Instead of the four theoretically possible phases of rhythmic neural activity, all experiments yielded recordings of biphasic activity, even in conditions of electrical stimululation of the cerebrobuccal connectives, which triggers rhythmic movements of this apparatus in preparations. These data demonstrate the phase-dependent coordination of two independent rhythmic food-procuring movements in Clione.

Synaptic contact between mechanosensory neuron and withdrawal interneuron in terrestrial snail is mediated by L-glutamate-like transmitter.

The properties of the monosynaptic input from mechanosensory neurons to withdrawal interneurons were examined in Helix lucorum. The instantaneous I-V relation of the excitatory postsynaptic current in withdrawal interneurons was nonlinear, having a plateau region between -40 and -60 mV. On application of the blocker of vertebrate N-methyl-D-aspartate (NMDA) receptors AP5, or reduction of the Mg(2+) concentration, the current-voltage relation became more linear, suggesting that Mg(2+) may partially block the ion channel underlying the EPSC at voltages ranging from -40 to around -60 mV and the involvement of NMDA-like receptors. DNQX and 6-cyano-7-nitroquinoxaline-2,3-dione, which are known to block the glutamate non-NMDA receptors in mammals, significantly depress in a dose-dependent manner the actions of the natural transmitter. Exogenous L-glutamate applications mimicked the action of the mechanosensory neuron transmitter.

Participation of GABA in establishing behavioral hierarchies in the terrestrial snail.

GABA-immunoreactive fibers were observed in the neuropile of each ganglion of Helix lucorum, while GABA-immunoreactive neural somata were found only in the buccal, cerebral, and pedal ganglia. Bath application of 10(-5) M GABA to the preparation "buccal mass-buccal ganglia" elicited a sequence of radula movements characteristic of feeding behavior. Corresponding bursts of activity were recorded in the buccal nerves under GABA application and in the buccal neurons recorded optically. In preparations of isolated central nervous system, the bath applications of GABA (10(-5) to 10(-4) M) elicited no changes in synaptic input of the premotor interneurons involved in the withdrawal behavior. However, a significant decrease in amplitude of the synaptic input and in the number of spikes in responses elicited by the test nerve stimulation was observed in metacerebral serotonergic neurons involved in modulating the feeding behavior. GABA application inhibited the spontaneous spike activity in some pedal serotonergic neurons involved in the network underlying withdrawal responses and evoked bursting activity in the other neurons of this functional group. The effects of GABA application on mechanically isolated serotonergic neurons suggest that the primary effect of GABA is inhibition. Thus, our results give evidence of the putative role of GABA in activating the feeding behavior and in the synergistic suppression of serotonergic modulation of the withdrawal behavior and serotonergic modulation of feeding, which has corresponded to the observed behavioral suppression of withdrawal reactions during feeding.

A single serotonergic modulatory cell can mediate reinforcement in the withdrawal network of the terrestrial snail.

A cluster of 40 serotonergic cells in the rostral part of pedal ganglia of the terrestrial snail Helix lucorum was shown previously to participate in the modulation of withdrawal behavior and to be necessary during the acquisition of aversive withdrawal conditioning in intact snails. Local extracellular stimulation of the serotonergic cells paired with a test stimulus elicited a pairing-specific increase (the difference between paired and explicitly unpaired sessions was significant, p <.01) of synaptic responses to test stimulation in the premotor interneurons involved in withdrawal. This result suggested participation of serotonergic cells in mediating the reinforcement in the withdrawal network. Intracellular stimulation of only one identified Pd4 cell from the pedal group of serotonergic neurons paired with a test stimulus also significantly increased (the difference between paired and explicitly unpaired sessions was significant, p <.05) synaptic responses to paired nerve stimulation in same premotor interneurons involved in withdrawal. Morphological investigation of a cluster of pedal serotonergic neurons showed that only the Pd4 cell had branches in the parietal ganglia neuropile where the synapses of premotor withdrawal interneurons and of presynaptic neurons are located. The data suggest that a single serotonergic cell can mediate the reinforcement in the withdrawal network of the terrestrial snail. Patterns of responses of the Pd4 cells to tactile and chemical stimuli conform to the suggestion.

[Phase-dependent coordination of two motor programs in the buccal ganglion of a pteropod mollusk]. / Fazozavisimaia koordinatsiia dvukh motornykh programm v bukkal'nom ganglii krylonogogo molliuska.

Rhythmic activities of two feeding structures of the pteropod mollusk Clione limacina, redula and hooks, controlled by the neural networks in the buccal ganglia must be coordinated in order to produce a meaningful feeding response. Optical recording from the buccal ganglia, which allows the simultaneous activities of numerous neurons to be traced, revealed that such coordination exists in a phase-dependent manner. Instead of recording four theoretically possible phases of neuronal rhythmic activity, we always recorded only two phases, even after the electrical stimulation of the cerebro-buccal connective, which triggers both radula and hook rhythmic movements in the preparation.

Two modulatory inputs exert reciprocal reinforcing effects on synaptic input of premotor interneurons for withdrawal in terrestrial snails.

A cluster of serotonergic cells in the rostral part of pedal ganglia of the terrestrial snail Helix lucorum was shown previously to participate in modulation of withdrawal behavior, and to be necessary for elaboration of aversive withdrawal conditioning in intact snails. In the present experiments local extracellular stimulation of the serotonergic cells elicited a pairing-specific increase (difference between paired and explicitly unpaired sessions was significant, P<0.01) of synaptic responses in the premotor interneurons involved in withdrawal to paired nerve stimulation. Intracellular stimulation of only one Pd4 cell from the pedal group of serotonergic neurons increased (P<0.05) synaptic responses to contingent test nerve stimulation significantly in the same premotor interneurons for 2-3 hr. Mesocerebral cells are known to participate in male sexual behavior, and their extracellular stimulation was shown previously to suppress the amplitude of synaptic responses in withdrawal interneurons. Local extracellular stimulation of the mesocerebral cells elicited a pairing-specific decrease (P<0.01) of synaptic responses to contingent test nerve stimulation in the premotor interneurons involved in withdrawal for 2-3 hr. Paired application of met-enkephaline (10(-6) M, some mesocerebral cells are enkephaline-like immunoreactive) also selectively decreased synaptic responses to contingent nerve stimulation in the premotor interneurons for hours. Thus, two modulatory inputs exert pairing-specific effects that influence the same synaptic connection in opposite directions, which may underlie the long-term up- and down-regulation of behavioral responses.

Effects of serotonin levels on postsynaptically induced potentiation of snail neuron responses.

Studies on identified neurons in the common snail were performed to investigate potentiation of EPSP arising after intracellular tetanization of the post-synaptic neuron. These experiments showed that high-frequency intracellular tetanization of a command neuron leads to biphasic long-term increases in the amplitude of synaptic responses to test stimulation. The role of serotonin in forming potentiation was studied. It was suggested that the presence of particular serotonin concentrations in the intercellular fluid is required for forming the second phase of the increase in synaptic responses, while the first (transient) phase is insensitive to CNS serotonin levels.

[The effect of the serotonin level on the postsynaptically induced potentiation of neuronal responses in the snail]. / Vliianie urovnia serotonina na postsinapticheski indutsirovannuiu potentsiatsiiu otvetov neironov ulitki.

The EPSP potentiation induced by the intracellular tetanization of a postsynaptic neuron was studied in the identified neurons of a terrestrial Helix lucorum snail. The high-frequency intracellular tetanization of the command neuron induced a two-phase long-term increase in the amplitude of synaptic responses. The role of 5-HT was studied in potentiation formation. The obtained evidence suggests that the presence of a certain 5-HT concentration in the extracellular fluid is necessary for the second phase formation while the first (short-term) phase is insensible to the 5-HT level in the CNS.

Postsynaptic induction of long-term synaptic facilitation in snail central neurones.

Long-term facilitation in molluscs is believed to be induced due to purely presynaptic activations. We recorded excitatory postsynaptic potentials (EPSPs) simultaneously from two identified neurones of snail parietal ganglia. We report a non-decrementing facilitation induced by intracellular tetanization with concomitant presynaptic activation. The mean EPSP amplitude measured in 10 neurones 30-50 min after tetanization was 17% greater than in the non-tetanized control neurones. Only short-lasting (5-10 min) postsynaptic changes were found (post-tetanic hyperpolarization and resistance decrease). The facilitation was especially prominent (34%, 10 min post-tetanus) but decreased within 15 min in preparations with rapid wash-out of the external media. The data suggest that induction of long-term enhancement in molluscs depends on postsynaptic events and, like in mammals, may involve increased postsynaptic Ca2+ and subsequent release of retrograde messengers.

Participation of cardioactive peptides in habituation and sensitization of the synaptic input of command neurons of snail defense behavior.

The influence of serotonin, a small cardioactive peptide (SCPb) and FMRFamide on the time course of the responses of defense behavior command neurons in response to rhythmic stimulation of the intestinal nerve was studied in a preparation of the isolated common snail nervous system. It was found that the application of serotonin and SCPb induces an increase in the absolute magnitude of the synaptic response of the neurons under investigation; however, testing of the time course of the responses against the background of the action of these substances reveals the absence of sensitization and an increase in the rate of habituation to the rhythmic stimulus. The effect of the action of FMRFamide was entirely opposite to the effect of serotonin and SCPb, in both the influence on the amplitude of the single response and on the rate of habituation of the responses of the neurons. The data obtained point to the independence of the subcellular mechanisms of sensitization and habituation.

[The participation of cardioactive peptides in the processes of habituation and sensitization of the synaptic input to the command neurons in snail defensive behavior]. / Uchastie kardioaktivnykh peptidov v protsessakh privykaniia i sensitizatsii sinapticheskogo vkhoda komandnykh neironov oboronitel'nogo povedeniia ulitki.

Small cardioactive peptide (SCPb) application in a concentration of 5 x 10(-8) M in the saline results in a significant increase of the amplitude of the summary excitatory postsynaptic potential elicited by the intestinal nerve stimulation in the command neurons (CN) for withdrawal reactions. Serotonin (5-HT) exerts comparable effects in a concentration of 10(-6) M. FMRFamide application (10(-6) M) significantly decreases the amplitude of the synaptic response to a single stimulus. Habituation rate of synaptic responses to the rhythmic (0.1 cps) stimulation in CN is significantly increased by serotonin application. SCPb acts in a similar fashion but in a lower concentration. FMRFamide application in a concentration of 10(-6) M affects in the opposite way the habituation rate of synaptic responses to rhythmic stimulation and in some cases evokes sensitization of responses.

The neurochemical basis of backward inhibition in a defense reaction reflex arc.

An investigation was carried out on a preparation of the isolated nervous system of the edible snail of the mechanism of habituation in the network of defense behavior neurons. It was established that the intracellular activation of one of the systems of the defense behavior command neurons, as in the case of the application of the neuropeptide FMRF-amide, leads to a decrease in the amplitude of the postsynaptic potentials in the remaining neurons of this class. An inference is reached, when account is taken of the presence of endogenous FMRF-amide in the defense behavior command neurons, regarding the possibility of active inhibition of the behavioral response to an appropriate stimulus through this neurochemical mechanism of the backward connection. The possible interrelationships with other modulating systems and the significance of such an association in the organization of behavior are discussed.